Early in embryonic development, one of the two X chromosomes in mammalian females becomes heterochromatic, late replicating, and largely genetically inactive as a means of dosage compensation for X-linked genes. Once established, this inactivation is irreversible and clonally stable in somatic cells. While the basic features of X inactivation have been well established for some time, the molecular basis for the initiation and promulgation of this chromosome-based cis-regulatory signal remains unknown. The proposed research will address a number of key questions regarding fundamental aspects of the X inactivation process. How does the process initiate early in embryogenesis? Are one or more X inactivation centers required for initiation? If a specific locus is required for initiation of X inactivation, is it also required for the maintenance of inactivation through successive cell divisions? What proportion of genes are subject to inactivation and what proportion "escape" inactivation? Are inactivated and non-inactivated genes clustered in specific regions of the chromosome or is X inactivation controlled on a locus basis? How is it determined which genes respond to and which genes are refractory to the X inactivation signal? Our analysis of human X chromosome inactivation will focus initially on the genetic basis for the initiation and maintenance of X inactivation and on the nature of genes that escape inactivation. The specific aims of the proposed research are: (i) to examine the transcriptional basis for inactivation of X-linked genes in a series of human/mouse somatic cell hybrids retaining either "active" X or "inactive" X chromosomes; (ii) to identify and characterize new genes that escape the inactivation process by isolating human cDNA clones expressed from "inactive" human X chromosomes; (iii) to test the hypothesis that there is a single region on the X responsible for X inactivation by examining structurally abnormal inactive X chromosomes in somatic cell hybrids to define the putative X inactivation center; (iv) to evaluate whether the continued presence of such a locus is required for the stable maintenance of the inactive X; (v) to isolate the X inactivation center in yeast artificial chromosomes; and (vi) to establish a system to examine human X chromosome and/or yeast artificial chromosomes carrying large fragments of DNA including candidate sequences for the human X inactivation center.